Molecular Identification of Bacteria Isolated from Marketed Sparus aurata and Penaeus indicus Sea Products: Antibiotic Resistance Profiling and Evaluation of Biofilm Formation

Background: Marketed fish and shellfish are a source of multidrug-resistant and biofilm-forming foodborne pathogenic microorganisms. Methods: Bacteria isolated from Sparus aurata and Penaeus indicus collected from a local market in Hail region (Saudi Arabia) were isolated on selective and chromogenic media and identified by using 16S RNA sequencing technique. The exoenzyme production and the antibiotic susceptibility patterns of all identified bacteria were also tested. All identified bacteria were tested for their ability to form biofilm by using both qualitative and quantitative assays. Results: Using 16S RNA sequencing method, eight genera were identified dominated by Vibrio (42.85%), Aeromonas (23.80%), and Photobacterium (9.52%). The dominant species were V. natrigens (23.8%) and A. veronii (23.80%). All the identified strains were able to produce several exoenzymes (amylases, gelatinase, haemolysins, lecithinase, DNase, lipase, and caseinase). All tested bacteria were multidrug-resistant with a high value of the multiple antibiotic index (MARI). The antibiotic resistance index (ARI) was about 0.542 for Vibrio spp. and 0.553 for Aeromonas spp. On Congo red agar, six morphotypes were obtained, and 33.33% were slime-positive bacteria. Almost all tested microorganisms were able to form a biofilm on glass tube. Using the crystal violet technique, the tested bacteria were able to form a biofilm on glass, plastic, and polystyrene abiotic surfaces with different magnitude. Conclusions: Our findings suggest that marketed S. aurata and P. indicus harbor various bacteria with human interest that are able to produce several related-virulence factors.


Introduction
Seafood has great nutritional benefits and economic importance; thus, the bacterial species present in seafood must be identified and studied to determine the best health practices to prevent seafood-borne illnesses [1]. Fish is the food category mainly associated with foodborne outbreaks, accounting for approximately 6-8% of the total food-borne diseases. This prevalence is greater than the incidence of food illness cases from chicken and beef [2]. Both pathogenic and harmful bacteria can be introduced into seafood products during the manufacturing process and in the supply chain [3,4]. Shellfish are considered as a major source of seafood-borne pathogens in humans, as they are usually consumed undercooked or raw. Water warming due to climate change has recently become an issue as it would elevate the microbial population, including Vibrio species in particular foodborne strains and other pathogenic bacteria that ultimately end up in seafood environments, inducing more denaturing step for 5 min, 30 cycles of amplification (30 s denaturation at 95 • C, 30 s annealing step at 52 • C, and 30 s extension at 72 • C), and a final extension step at 72 • C for 5 min. After PCR, the obtained products of the 27F-907R regions were electrophoresed using 2% agarose gel (110 V, 150 mA, 45 min). It was observed that the size of the products was between 1200-1400 bp. Then, the cleanup phase was started using ExoSAP-IT Express PCR Cleanup Reagents. For the experiment, 10 µL of each PCR product was mixed with 4 µL of the cleanup reagent.
Molecular identification of isolated bacteria was achieved by sequencing the 16S rDNA gene using the universal primer 27F (5 -AGAGTTTGATCMTGGCTCAG-3 ) and the reverse primer 907R (5 -CCGTCAATTCCTTTRAGTTT-3 ) previously described by Muyzer et al. [19]. After the DNA sequencing reaction was completed, the sequencing products were purified using the gel filtration method with Sephadex. After the purification process, the DNA sequencing process was started. This was performed on the ABI 3130XL device using the capillary electrophoresis method.
Sequence alignment was performed using BioEdit version 7.1.3.0 [20], and a total of 859 bp were successfully aligned in the final dataset consisting of 31 nucleotide sequences, including Salmonella enterica as the outgroup. The 16S rDNA sequences were subjected to a BLAST search to determine the sequence homology with the sequences previously deposited in the NCBI to identify isolated bacterial species and strains. The sequences with the highest homology belonging to Vagococcus fluvialis, Staphylococcus aureus, Staphylococcus epidermidis, Shewanella indica, Photobacterium damselae, Morganella morganii, Bacillus cereus, Aeromonas veronii, and Vibrio harveyi were added to our dataset to determine the relation of our isolates with them. A phylogenetic tree was generated by the neighborjoining method [21] with a bootstrap test (1000 replicates) [22]. The Kimura two-parameter method [23], the best-fitting model for our sequence dataset, was used to compute the pairwise evolutionary distances among the sequences, with the gaps removed by the pairwise deletion option implemented in MEGA 11 software [24].

Exoenzyme Production
The identified isolates were tested for their abilities to produce several exoenzymes including DNAase, lipase, amylase, caseinase, and lecithinase, according to the protocol described by Hörmansdorfer et al. [25] and Snoussi et al. [26]. For the experiment, PBS agar medium was supplemented with Tween-80 (lipase activity), starch (amylase activity), skim milk powder (caseinase activity), and egg yolk (lecithinase production). Petri dishes were incubated for 72 h at 37 • C, and the formation of a clear zone around the inoculated spots was considered a positive test. In addition, hemolysin production was tested on human blood agar supplemented with 5% human blood (Oxoid Ltd., Basingstoke, UK) [26].
Two mathematic indices were used to interpret the results obtained: (i) the antibiotic resistance index (ARI) of each bacterial population [30], and (ii) the multiple antibiotic resistances (MAR) index of the isolates [31].

Adhesion Properties and Biofilm Formation Screening
The ability of all identified bacteria to secrete an exopolysaccharide layer (slime production) was tested using the same protocol previously described by Snoussi et al. [32] adapted to Vibrio species. Colonies obtained on Congo red agar were interpreted as slime producers (pigmented colonies), while unpigmented colonies were interpreted as slime nonproducers [33].

Wolfe Test
The ability of the identified bacteria to adhere to the glass surface was tested using the same protocol described by Wolfe et al. [34]. For the experiment, a 10 mL glass tube (0.5 cm in diameter) containing seawater broth (5 g of Bactotryptone, 3 g of yeast extract, and 3 mL of glycerol in 700 mL of seawater and 300 mL of purified water) was used to grow overnight all bacteria at 37 • C. Afterward, 100 µL of this pre-enriched culture were added to inoculate new tubes containing the same medium and incubated at 37 • C for 10 h without shaking. Following a 15 min staining period with 1% (w/v) crystal violet, all glass tubes were cleaned with distilled water before being used for further testing. Glass-biofilm positives were bacteria that produced a purple pellicule on the cultures' air surface.

Biofilm Formation on Glass and Plastic Surfaces
Glass material (circular 12 mm diameter cover glasses) and a plastic surface (12 mm diameter) were used for the quantitative estimation of biofilm-forming capacities of all identified strains from S. aurata and P. indicus inserted into the bottom of 24-well (15 mm diameter each well) microtiter plates and filled with 2 mL of each bacterial suspension (10 9 UFC/mL in PBS) for 24 h at 37 • C. The experiments were carried out in triplicate and three times. The same procedure described by Henriques et al. [36] was followed using 600 µL of crystal violet for 5 min to stain the biofilm-forming bacteria fixed on the abiotic surfaces selected. The pieces were gently washed in water and dried before being immersed in 1 mL of 33% (w/v) acetic acid to release and dissolve the stain. Using a microtiter plate reader, the OD of the resulting solution was measured at 570 nm (Bio-Tek, Model Synergy HT, city). Results were interpreted using the scheme proposed by Stepanović et al. [37], where bacteria were interpreted as nonadherent (0) OD ≤ ODc, weakly adherent (+) ODc < OD ≤ 2 × ODc, moderately adherent (++) 2 × ODc < OD ≤ 4 × ODc, or strongly adherent (+++): 4 × ODc < OD.

Statistical Analysis
All experiments were performed in triplicate, and the average and standard deviation were calculated using the SPSS 25.0 statistical package for Windows.

Morphological Characterization and 16SRNA Identification of Bacterial Isolates
The analysis of different samples from P. indicus on TCBS agar medium revealed the characterization of two morphotypes: yellow colonies of 1 to 2 mm in diameter and green-yellow colonies with a diameter of about 2-3 mm, respectively. In addition, on Vibrio ChromoSelect agar, four different morphotypes based on color and size were observed (Table 1). Similarly, 14 morphotypes were obtained from S. aurata after being cultured on TCBS and Vibrio ChromoSelect agar medium. The dominant color on TCBS agar plates was yellow (diameter between 1 and 7 mm). However, on Vibrio ChromoSelect agar, nine isolates with various ranges of colony shapes, sizes, and colors were seen, including blue, turquoise, purple, pink, light green with a green center, and colorless colonies ( Table 1). Twenty-one colonies were subsequently analyzed using molecular techniques (16S RNA) and bioinformatics (BioEdit software and National Center for Biotechnology Information (NCBI)) to certain the identity of these isolates. The results demonstrated that the main bacteria identified in both samples belonged to the Vibrio genus with four different species, including V. natriegens, V. harveyi, V. alginolyticus, and V. hyugaensis. The second most dominant species was Aeromonas veronii. The phylogenetic tree ( Figure 1) shows the genetic relationship of the 21 identified bacterial strains, isolated from various organs of S. aurata and shrimps (P. indicus) using the NJ method. Twenty-one colonies were subsequently analyzed using molecular techniques (16S RNA) and bioinformatics (BioEdit software and National Center for Biotechnology Information (NCBI)) to certain the identity of these isolates. The results demonstrated that the main bacteria identified in both samples belonged to the Vibrio genus with four different species, including V. natriegens, V. harveyi, V. alginolyticus, and V. hyugaensis. The second most dominant species was Aeromonas veronii. The phylogenetic tree ( Figure 1) shows the genetic relationship of the 21 identified bacterial strains, isolated from various organs of S. aurata and shrimps (P. indicus) using the NJ method.

Exoenzyme Production
The bacteria identified in this study were investigated for their capability to produce several hydrolytic enzymes. The results demonstrated that all tested bacteria were able to produce amylase (100%), but other enzymes were only induced by some bacteria. The percentage of the total bacteria secreted by each enzyme was as follows: lipase (80.95%),

Exoenzyme Production
The bacteria identified in this study were investigated for their capability to produce several hydrolytic enzymes. The results demonstrated that all tested bacteria were able to produce amylase (100%), but other enzymes were only induced by some bacteria. The percentage of the total bacteria secreted by each enzyme was as follows: lipase (80.95%), DNase (71.42%), caseinase (66.66%), lecithinase (57.14), hemolysins (52.38), and gelatinase (47.61%) ( Table 2). Some bacteria were able to produce all enzymes tested, such as V. harveyi (P9), V. alginolyticus (P2), A. veronii (SA15), V. fluvialis (SA21). It was observed that some bacteria with the same identify had different exoenzyme profiles.
In addition, it is worth noting that Vibrio spp. (n = 9) were particularly completely resistant to ceftaroline, tigecycline, ticarcillin, colistin, and meropenem ( Figure 3). On the other hand, these bacteria were found to be sensitive or dose-dependently resistant (intermediate) to netilmicin, norfloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole. Similarly, the Aeromonas spp. (n = 5) strains were completely resistant to amikacin, moxifloxacin, ceftaroline, tigecycline, amoxicillin-clavulanic acid, ampicillin, ticarcillin, and meropenem. The lowest percentage of resistance was recorded with three antibiotics (tobramycin, norfloxacin, and trimethoprim-sulfamethoxazole) ( Figure 3). In addition, it is worth noting that Vibrio spp. (n = 9) were particularly completely resistant to ceftaroline, tigecycline, ticarcillin, colistin, and meropenem ( Figure 3). On the other hand, these bacteria were found to be sensitive or dose-dependently resistant (intermediate) to netilmicin, norfloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole. Similarly, the Aeromonas spp. (n = 5) strains were completely resistant to amikacin, moxifloxacin, ceftaroline, tigecycline, amoxicillin-clavulanic acid, ampicillin, ticarcillin, and meropenem. The lowest percentage of resistance was recorded with three antibiotics (tobramycin, norfloxacin, and trimethoprim-sulfamethoxazole) (Figure 3). Overall, the tested bacteria could be considered as multidrug-resistant microorganisms, as all isolates were resistant to three or more antibiotics from different classes (Supplementary Tables S1 and S2  Overall, the tested bacteria could be considered as multidrug-resistant microorganisms, as all isolates were resistant to three or more antibiotics from different classes (Supplementary  Tables S1 and S2). In fact, the multiple antibiotic resistance index (MARI) for Vibrio spp. (n = 9) ranged from 0.384 (V. harveyi P9) to 0.653 (V. alginolyticus P2). Regarding the Aeromonas spp. strains (n = 5), the MARI ranged from 0.461 (A. veronii SA17) to 0.692 (A. veronii SA25). The MARI ranged from 0.423 (M. morganii P5) to 0.653 (S. indica P13) for the other Gram-negative identified bacteria. Similarly, for Gram-positive bacteria, the MARI was about 0.5 for B. cereus SA9, 0.333 for S. epidermidis SA7, and 0.277 for V. fluvialis SA21. In addition, our results revealed that the calculated ARI varied from 0.542 for all Vibrio strains (n = 9) to 0.553 for all Aeromonas strains (n = 5). Taken together, the ARI for the 18 Gram-negative bacteria was about 0.544, while the same index was lower (ARI = 0.462) for the Gram-positive bacteria tested (Table 3). Table 3. Distribution of ARI in the different bacterial populations identified in this study.

Slime Production on CRA Plates and Glass Tubes (Wolfe Test)
The phenotypic production of slime was assessed by culturing the bacteria on Congo Red Agar plates. Pigmented colonies were considered as normal slime-producing bacteria, whereas colorless colonies were classified as non-slime-producing. Among the tested isolates, six out of 21 (28.57%) were able to induce slime, indicated by black colonies, and the remaining 15 bacteria were non-slime-producing characterized by red, orange, Bordeaux, white with a red center, or black-gray morphotypes (Figure 4).

Slime Production on CRA Plates and Glass Tubes (Wolfe Test)
The phenotypic production of slime was assessed by culturing the bacteria on Congo Red Agar plates. Pigmented colonies were considered as normal slime-producing bacteria, whereas colorless colonies were classified as non-slime-producing. Among the tested isolates, six out of 21 (28.57%) were able to induce slime, indicated by black colonies, and the remaining 15 bacteria were non-slime-producing characterized by red, orange, Bordeaux, white with a red center, or black-gray morphotypes (Figure 4). All tasted bacteria were able to adhere to the glass, giving a purple pellicule on the air surface of the glass tube, except for Photobacterium damselae. The intensity of the color of the crust formed ranged from intense to moderate. Moreover, 10 bacteria out of 21 tested were strongly adhesive to glass surface ( Figure 5). All these data are summarized in Table 4.   All tasted bacteria were able to adhere to the glass, giving a purple pellicule on the air surface of the glass tube, except for Photobacterium damselae. The intensity of the color of the crust formed ranged from intense to moderate. Moreover, 10 bacteria out of 21 tested were strongly adhesive to glass surface ( Figure 5). All these data are summarized in Table 4.
More recently, Beyari and colleagues [49] studied the bacterial diversity in some marketed fish retails from Jeddah province and reported the identification of 17 different bacterial genera (dominated by Aeromonas, Pseudomonas, Psychrobacter, and Alcaligens). The same authors reported the identification of 32 different species including some human pathogenic ones such as R. aquatilis, Proteus vulgaris, Klebsiella quasipneumoniae, Yersinia enterocolitica, P. lundensis, P. oryzihabitans, Psychrobacter phenylpyruvicus, P. sanguinis, Alcaligenes faecalis, and P. putida.
The presence of pathogenic bacteria in fish and other seafood is thought mainly to result from the growth conditions, harvesting, and preservation processes that support the spread of microorganisms, particularly pathogens. Therefore, it has been found that cautious processing methods could significantly reduce microbes in fish and other seafood [51]. The major isolated bacteria found in the study were Gram-negative bacteria. This result is similar to previous publications [52]. The main bacteria identified in the current study were Vibrio species, accounting for 43% of the total bacteria identified. Vibrio species, including V. harveyi, V. alginolyticus, V. natriegens, and V. hyugaensis are associated with many human and fish diseases. The next most abundant genus was Aeromonas veronii in both fish and shrimp; this bacterium, along with other Aeromonas species, has been linked to diarrhea cases in children [53], where approximately 8% of acute enteric infections are induced by Aeromonas species [54]. It was found that most Aeromonas species could be isolated from different environments, including rivers, meat, and fish, as well as from patients suffering from diarrhea [55][56][57].
Thus, Aeromonas species are considered to be primary pathogens in aquaculture that can grow at refrigerator temperature and, hence, can be a major source of food contamination, especially where is a probability of cross-contamination with preparedto-consume food products [58]. Recently, many fish infections have been initiated by Aeromonas species [59][60][61][62]. Other bacterial genera detected in this study include Shewanella, Photobacterium, Vagococcus, Staphylococcus, and Bacillus. Bacteria can live comfortably in aquatic settings; thus, bacterial infection has become the main barrier to the success of aquaculture farms [63].
This investigation also looked at the ability of the identified bacteria to produce extracellular enzymes, which have been recognized as an indicator of health risk in microbes isolated from different sources, including clinical, food, and environmental samples [64,65]. It was found that the bacterial isolates could yield at least two exoenzymes, including amylase, the only enzyme was produced by all isolates. These results indicated that all isolated bacteria produced a variety of extracellular enzymes, but each isolate had a distinct pattern of hydrolytic enzymes. Enzymes produced by bacteria could potentially modulate the bacterial virulence and pathogenicity, breaking down proteins and making them available for proliferation [66,67]. The secretion of some enzymes and toxins has been found to be responsible for food spoilage and can make the bacteria more resistant to antibiotic agents, leading to therapeutic issues [68]. Amylase was the only enzyme produced by all isolated bacteria, which may indicate the capability of all isolates to use this enzyme to hydrolyze starch [69]. Almost 50% of Vibrio isolates in the current study expressed all exoenzymes tested, with complete production of lipase and amylase. In a similar manner, several extracellular enzymes were produced by Vibrio [33,68]. Approximately half of the bacterial isolates were capable of producing hemolysin and gelatinase; these enzymes are recognized as virulence factors as both are associated with bacterial pathogenicity [70]. In addition, 80% of the isolates had lipolytic activity, which is associated with the acquisition of nutrients by degrading host lipids. More than 66% of the isolates had DNase and caseinase activities. DNase has a function as an endonuclease and partially plays a role in DNA hydrolysis, whereas caseinase is associated with bacterial pathogenicity. Several exoenzymes have been detected in Vibrio bacteria from different sources, including fish, shrimp, and shark [71,72].
The result of phenotypic slime production revealed that 28% of the total isolates were positive slime producers. These isolates belong to the Vibrio and Anemones genera. Slime production is measured as an important virulence factor in some pathogenic bacteria, including Vibrio and Aeromonas species, and it could be an indicator of a high-risk commination [75]. Slime is used by bacteria as a protective mechanism against external environments; thus, microbes coated with slime are more resistant to antibiotics and other stressors. Slime molecules are considered to be involved in biofilm formation; indeed, they play a significant role in the initial stages of biofilm development [76,77]. Previous studies have reported the characterization of several morphotypes formed by V. alginolyticus isolated from fish (S. aurata, D. labrax) on Congo red agar, and most of them were slime producers with black colonies [32,33]. Similarly, Snoussi and colleagues [16] reported the identification of A. hydrophila, Staphylococcus spp., V. alginolyticus, Enterobactercloacae, K. ornithinolytica, K. oxytoca, and Serratia odorifera from seabass, seabream, roseshrimp, and blue mussel. These strains produced five morphotypes based on the colorimetric scale on the tested medium (Bordeaux, red with dark center, pink with red center, pink, and red colonies).
The capacity of the isolates to form biofilms on different materials, including polystyrene, glass, and plastic, was investigated. The analysis indicated that 42.8% of the isolates formed biofilms on polystyrene, in contrast to 90.4% on glass and 85.7 on plastic, to varying degrees ranging from weak to strong. Biofilm development seemed to be affected by surface properties; the use of polystyrene materials is highly recommended to avoid biofilm formation [78]. Some isolates, including V. alginolyticus, A. veronii, P. piscicida, and B. cereus, tended to form biofilms on all tested surfaces. The results of this study are similar to others demonstrating that Aeromonas and Vibrio species from fish and shellfish and their surrounding water were able to form biofilms on different biotic and abiotic surfaces to different degrees [32,33,79]. Bacteria that develop biofilms are greatly resistant to changing environments, including antibiotics and detergents [80,81]. Thus, microbial biofilm development is a topic of important interest in many fields, including food and medical industries, as it is a significant contributor to bacterial virulent, which can lead to serious infections that are difficult to treat [82,83]. The precipitation of mineral and food residues in food manufacturing could positively affect the development of biofilms [84].

Conclusions
This investigation provided clear evidence that both fish and shrimp collected from local markets, having been initially harvested from an aquaculture farm, had a diversity of bacterial genera. The outcomes of this study revealed that the main bacterial genera identified were Vibrio and Aeromonas. Antimicrobial resistance was also demonstrated in all bacterial isolates, and high multidrug resistance indices were obtained in most of the tested isolates. The majority of isolates were biofilm producers, suggesting a significant threat from these isolates in the food industry. Therefore, control and prevention of microbial contamination must be taken into consideration in order to obtain healthy and uncontaminated food, particularly seafood.